Development And Application Of Yolk Antibody Against Porcine Epidemic Diarrhe Virus

Porcine epidemic diarrhea virus（PEDV）is a highly infective intestinal pathogen of pigs.The infected pigs mainly have acute watery diarrhea,vomiting,dehydration and other clinical symptoms at different ages.Infection can occur in pigs,especially in suckling piglets.The PEDV epidemic has continued to occur in most pig-producing Asian countries.Since2010,I have had PEDV variants.Although the pigs were previously immunized with the CV777 vaccine,the mutant strains have caused huge economics in our pig industry loss.At present,there is no effective treatment after PEDV infection.Antibiotics,Chinese herbal medicines and antiviral therapy are not effective,and there are large side effects.The yolk antibody has the excellent characteristics of prevention and treatment of the virus and is confirmed and accepted by the researcher.Immunoglobulin of yolk（IgY）is an immunoglobulin extracted from egg yolk produced by chickens immunized with a specific pathogen.An environmentally friendly biological preparation with strong specificity,no toxic side effects,no toxic residue,and no drug resistance.In this experiment,the isolated PEDV was inactivated to prepare immunogenic immunized laying hens,and a high concentration of anti-PEDV yolk antibody was extracted from egg yolk,and the traits and characteristics of the extracted and purified yolk antibody were tested for the prevention and treatment of PEDV.The main findings are as follows:1.Collected 19 batches of suspected PEDV-positive small intestine samples with clinical symptoms of diarrhea,dehydration and vomiting in 10 large-scale pig farms in Henan Province from 201 to 2018.The genome of the disease was analyzed by PCR.Detection of PEDV pathogens.Among them,the PEDV positive disease material was 13batches,and the total batch positive rate reached 68.4%.2.Virus isolation was performed on the detected PEDV-positive disease material,and typical PEDV cytopathic lesions appeared in Vero cells after 7 samples were poisoned.The isolated PEDV strain was inoculated again with Vero cells for IFA identification.The results showed that Vero cells inoculated with PEDV group showed specific fluorescence.Seven strains of PEDV virus were successfully isolated.Five strains of PEDV virus strains were tested for virus toxicity.The results showed that the PEDV strain was between 103.5-106.0TCID50/mL,the PEDV S gene was amplified,and the virus S gene was sequenced and analyzed.The homology analysis and phylogenetic tree construction of the S gene were analyzed.It was concluded that the S gene of PEDV171120 strain was inserted and belonged to the current epidemic strain,which was inactivated as the egg chicken immunogenic strain.3.The yolk antibody in egg yolk was separated and purified by water dilution-saturated ammonium sulfate method.The yolk antibody was concentrated by dialysis method,and the antibody protein was run by SDS-PAGE electrophoresis to show two bands of interest.There are clear heavy and light chain bands,and the yolk antibody concentration is up to 8.9 mg/mL.The titer of the egg yolk antibody is determined by a commercial kit,and the highest OD₄₅₀is 2.876 under the microplate reader.The purified yolk antibody has a higher concentration and activity.The PEDV neutralizing antibody was determined by the endpoint method,and the virus-diluted serum method was immobilized.The cell experiment showed that the IgG antibody had a neutralization effect at a 1:1024 dilution,and the IgY antibody had a neutralization effect at a 1:512 dilution.This indicates that the yolk antibody has strong neutralizing activity.4.The purified egg yolk antibody was applied to clinical trials,and the efficacy of prevention and protection of egg yolk antibody was examined by first attacking and then feeding the egg yolk antibody and feeding the egg yolk antibody first.The prevention efficiency of piglet epidemic diarrhea is 60%,and the cure rate is 80%.5.Clinical dissection was performed on experimental animals,and the content of virus in the duodenum,front jejunum,middle jejunum,end jejunum and ileum of piglets was determined by fluorescence quantitative method.The results showed that the content of virus in jejunum segment was the highest,and the content of virus in duodenum and ileum segment was lower.At the same time,immunohistochemical identification of each segment showed that jejunal segment virus colonized most,duodenum and ileum segment virus colonized less.